Target degradation specificity of phytoplasma effector phyllogen is regulated by the recruitment of host proteasome shuttle protein

Abstract Phytoplasmas infect a wide variety of plants and can cause distinctive symptoms including the conversion of floral organs into leaf‐like organs, known as phyllody. Phyllody is induced by an effector protein family called phyllogens, which interact with floral MADS‐box transcription factors (MTFs) responsible for determining the identity of floral organs. The MTF/phyllogen complex then interacts with the proteasomal shuttle protein RADIATION SENSITIVE23 (RAD23), which facilitates delivery of the MTF/phyllogen complex to the host proteasome for MTF degradation. Previous studies have indicated that the MTF degradation specificity of phyllogens is determined by their ability to bind to MTFs. However, in the present study, we discovered a novel mechanism determining the degradation specificity through detailed functional analyses of a phyllogen homologue of rice yellow dwarf phytoplasma (PHYLRYD). PHYLRYD degraded a narrower range of floral MTFs than other phyllody‐inducing phyllogens, resulting in compromised phyllody phenotypes in plants. Interestingly, PHYLRYD was able to bind to some floral MTFs that PHYLRYD was unable to efficiently degrade. However, the complex of PHYLRYD and the non‐degradable MTF could not interact with RAD23. These results indicate that the MTF degradation specificity of PHYLRYD is correlated with the ability to form the MTF/PHYLRYD/RAD23 ternary complex, rather than the ability to bind to MTF. This study elucidated that phyllogen target specificity is regulated by both the MTF‐binding ability and RAD23 recruitment ability of the MTF/phyllogen complex.


| INTRODUC TI ON
Plant pathogens establish colonization on their host plants by secreting dozens of effector proteins to manipulate host developmental processes and suppress immune responses (Hogenhout et al., 2009).Functional analysis of effector proteins provides valuable information to understand the infection mechanisms of plant pathogens.Effector genes are usually exposed to selective pressures from the host plant, located in highly variable regions on pathogen genomes and undergo genetic mutations that result in differences in activity and functional diversification among homologous effectors (Raffaele & Kamoun, 2012;Upson et al., 2018).For example, Bentham et al. (2021) reported that APikL2, an effector conserved among blast fungus isolates from different grass hosts, has different amino acid polymorphisms in accordance with the host plant species, one of which broadens the binding specificity of APikL2 to its target host proteins.Thus, studying the functional variation within an effector family provides valuable insights into the molecular mechanisms of effector target specificity and co-evolution with host plant species, contributing to a better understanding of the roles of the effector family in pathogenicity.
Phytoplasmas ("Candidatus Phytoplasma" spp.) are a group of plant-pathogenic bacteria in class Mollicutes that are associated with various diseases in more than 1000 plant species (Marcone, 2014).
Recently, phyllogen-mediated MTF degradation was demonstrated to occur through the following steps: the phyllogen interacts with a target MTF; the MTF/phyllogen complex binds directly to RAD23 without ubiquitin; RAD23 delivers the complex to the proteasome, resulting in ubiquitin-independent proteasomal degradation of the target MTF (Kitazawa et al., 2022).
Several studies have reported phyllogens with impaired phyllody-inducing activity and MTF degradation activity (Aurin et al., 2020;Iwabuchi et al., 2019Iwabuchi et al., , 2020;;Liao et al., 2019).All of these phyllogens have defects in MTF-binding affinity, and the residues responsible for interaction with MTFs are concentrated on one surface of the phyllogen structure, which is a putative MTFbinding interface (Kitazawa et al., 2023).Based on these findings, together with the fact that phyllogen binds only to specific MTFs to induce their degradation (MacLean et al., 2014), the target specificity of phyllogens has been postulated to be determined by their MTF-binding affinity.
Our previous study revealed sequence diversity among phyllogen homologues and classified the homologues into groups phyl-A to -D (Iwabuchi et al., 2020).Interestingly, although the examined phyllogens in the phyl-A, C and D groups induced similar phyllody phenotypes in plants, all phyl-B phyllogens were found to completely lack phyllody-inducing activity.Site-directed mutagenesis analyses revealed that a single amino acid substitution conserved in the phyl-B group abolishes the MTF-binding affinity of phyllogens, resulting in defects in MTF degradation activity.
The "Candidatus Phytoplasma oryzae" rice yellow dwarf strain (RYD phytoplasma) is one of the most important pathogens of rice, causing severe systemic symptoms such as leaf yellowing, severe stunting and excessive tillering (Jung et al., 2003;Maejima, Oshima, & Namba, 2014).Notably, RYD phytoplasma induces floral abnormality in rice ears, including spikelet sterility and reduced ear numbers (Komori, 1965), although phyllody symptoms have not been reported.The phyllogen from RYD phytoplasma (PHYL RYD ) is the only full-length phyllogen identified from cereal-infecting phytoplasmas (Iwabuchi et al., 2020;Zhu et al., 2017).Although PHYL RYD belongs to the phyl-A group, it has multiple unique polymorphisms compared with other members in this group (Iwabuchi et al., 2020).

Despite these intriguing characteristics, functional characterization
of PHYL RYD has not yet been performed.Therefore, in this study, we investigated the phyllody-inducing activity and related functions of PHYL RYD .
However, the residues considered to constitute the putative binding interface with MTF (Kitazawa et al., 2023;Tokuda et al., 2023) are conserved, except for the glutamine at position 83 (Figure 1a).
In silico structure prediction using ColabFold (Mirdita et al., 2022) suggests that despite the multiple polymorphisms, the putative secreted region of PHYL RYD consists of two α-helices, similar to other phyllogens (Figure 1b,c; Iwabuchi et al., 2019;Liao et al., 2019).The PHYL RYD -specific polymorphisms were not concentrated on either the putative MTF-binding interface (Kitazawa et al., 2023) or other specific regions (Figure 1b,c).
F I G U R E 1 PHYL RYD possesses multiple unique polymorphisms but retains the basic structure.(a) Protein sequence comparisons between PHYL RYD and other phyl-A group phyllogens with phyllody-inducing activity.Sequences were aligned using MEGA v. 10.1.8(Kumar et al., 2018) with the MUSCLE algorithm (Edgar, 2004).Light blue arrowheads indicate insertions and amino acid substitutions specific to PHYL RYD .The residues considered to constitute the putative MADS box transcription factor (MTF)-binding interface (Kitazawa et al., 2023;Tokuda et al., 2023) are surrounded in orange.Numbers indicate the amino acid number of the putative secreted region of PHYL RYD .The background colour indicates the percentage of amino acid similarity: black 100%; dark grey 80%; light grey 60%.(b) The predicted threedimensional structure of PHYL RYD .The structure of the secreted region of PHYL RYD was predicted using ColabFold (Mirdita et al., 2022).The PHYL RYD -specific polymorphisms are shown in light blue, and the residues involved in MTF-binding are shown in orange similarly as in (a).The predicted aligned error (PAE) is shown on the right.The predicted local distance difference test (pLDDT) score and the predicted TM (pTM) score were 85.6 and 0.69, respectively.(c) The crystal structure of PHYL OY (PDB ID: 6JQA; Iwabuchi et al., 2019).PHYL OY is one of the phyl-A group phyllogens with phyllody-inducing activity.The residues involved in MTF-binding are shown in orange.

| Phyllody-inducing activity of PHYL RYD
To examine the phyllody-inducing activity of PHYL RYD , it was expressed in Arabidopsis thaliana and Nicotiana benthamiana by a modified tobacco rattle virus (TRV)-based gene expression vector (Iwabuchi et al., 2019).PHYL OY , a well-studied phyl-A group phyllogen of the "Candidatus Phytoplasma asteris" onion yellows strain (Kitazawa et al., 2022;Maejima, Iwai, et al., 2014), was used as a positive control.In A. thaliana plants, PHYL OY induced phyllody and virescence of the flowers, as described previously (Iwabuchi et al., 2019); the sepals, petals and stamens were converted into green leaf-like organs and the pistil changed into a stem-like structure (Figure 2a, upper panels).In contrast, PHYL RYD -expressing plants exhibited much less severe phenotypes than PHYL OY -expressing plants; the sepals often became small, and the petals sometimes turned greenish.However, homeotic conversions of the stamens into leaf-like organs and the pistil into the stem-like structure were rarely observed.In N. benthamiana, although >95% of PHYL OY -expressing flowers exhibited malformation or phyllody, >85% of PHYL RYD -expressing flowers showed no abnormalities (Figure 2a, lower panels; Figure S2).The expression of PHYL RYD or PHYL OY in the flowers of each plant species was confirmed by reverse transcription (RT)-PCR of the phyllogen-insertion region in pTRV2 (Figure 2b).
These results indicated that the phyllody-inducing activity of PHYL RYD was compromised in A. thaliana and N. benthamiana, albeit not completely lost.

| MTF degradation activity of PHYL RYD
Phyllogens including PHYL OY degrade APETALA1 (AP1) and SEPALLATA (SEP) 1-4 of A. thaliana (A-and E-class MTFs, respectively) and defects in their MTF degradation activity cause the loss of phyllody-inducing activity (Iwabuchi et al., 2020).To clarify the molecular mechanism of the significant attenuation of the phyllody phenotype induced by PHYL RYD , the MTF degradation activity of PHYL RYD was compared with that of PHYL OY (Figure 3a).Myc-fused AP1 and SEP1-4 (myc-AP1 and myc-SEP1-4) were transiently expressed with and without 3FLAG-fused phyllogen (3FLAG-PHYL RYD and 3FLAG-PHYL OY ) in N. benthamiana leaves, and protein accumulation was analysed by immunoblotting.Co-expression of 3FLAG-PHYL RYD significantly decreased the accumulation of myc-AP1, myc-SEP1 and myc-SEP2 to levels comparable to those of PHYL OY , indicating that PHYL RYD has degradation activity for these MTFs (PHYL RYD -degradable MTFs).In contrast, degradation activity was much lower for myc-SEP3 and myc-SEP4 (non-PHYL RYD -degradable MTFs) (Figure 3a).
Because the natural host plant of RYD phytoplasma is rice (Oryza sativa), we speculated that PHYL RYD may have adapted to degrade MTFs of O. sativa (Oryza MTFs), rather than MTFs of A. thaliana (Arabidopsis MTFs).To test this possibility, the degradation activity of PHYL RYD for Oryza MTFs belonging to the AP1-like clade (OsMADS14, OsMADS15 and OsMADS18) and SEP-like clade (OsMADS5 and OsMADS8) was also evaluated (Figure 3b).In addition to PHYL RYD and PHYL OY , we used PHYL SY , a non-phyllody-inducing F I G U R E 2 Phyllody-inducing activity of PHYL RYD is compromised.(a) Floral phenotypes of phyllogen-expressing plants.Arabidopsis thaliana and Nicotiana benthamiana were infected with tobacco rattle virus (TRV) vector carrying either PHYL RYD , PHYL OY or no exogenous gene (empty).Sepals, petals, stamens and pistils are indicated as se, pe, st and pi, respectively.(b) Confirmation of phyllogen expression.Total RNA was extracted from the flowers and reverse transcription (RT)-PCR was conducted to amplify the insertion region in pTRV2.18S ribosomal RNA (rRNA) was also amplified as an internal control.PCR using distilled water (DW) as a template was performed for a negative control.
phyl-B group phyllogen of the "Candidatus Phytoplasma fragariae" strawberry yellows strain (Iwabuchi et al., 2020).The results showed that all the tested Oryza MTFs were degraded by PHYL OY , but not at all by PHYL SY , which is consistent with the findings of a previous study using Arabidopsis MTFs (Iwabuchi et al., 2020).Compared with PHYL OY , degradation activity of PHYL RYD was similar or slightly inferior for OsMADS14, OsMADS15, and OsMADS5, but significantly lower for OsMADS8 and OsMADS18.These results do not support the idea that PHYL RYD has evolved to efficiently degrade Oryza MTFs.Rather, it is likely that the range of floral MTFs degraded by PHYL RYD is narrower than that degraded by PHYL OY for both Arabidopsis and Oryza MTFs.No clear correlation was found between the phylogenetic relationship of MTFs and degradation activity by PHYL RYD (Figure S3).

| MTF-binding affinity of PHYL RYD
To investigate the molecular mechanism underlying the narrow range of MTFs degraded by PHYL RYD , the MTF-binding affinity of PHYL RYD was examined in yeast two-hybrid (Y2H) assays (Table 1 and Figure S4).
GAL4 activation domain (AD)-fused MTF and DNA-binding domain (BD)-fused phyllogen were co-expressed in yeast cells, and their interaction was evaluated in terms of yeast growth on selective media.
The results indicated that PHYL RYD bound to all the tested Arabidopsis MTFs to the same extent, regardless of the degradation activity.
Similarly, although PHYL RYD showed different degrees of binding affinity depending on the type of Oryza MTFs, these differences did not correlate with its MTF degradation activity (Table 1).This result was in contrast to previous reports that phyllogens lacking MTF degradation activity show impaired MTF-binding affinity (Iwabuchi et al., 2019(Iwabuchi et al., , 2020;;Liao et al., 2019).Furthermore, the interaction between MTF and PHYL RYD was evaluated in planta by co-immunoprecipitation assays (Figure 4).AP1 and SEP3 were used as representatives of PHYL RYD -degradable MTFs and non-PHYL RYD -degradable MTFs, respectively.We co-expressed 3FLAG-fused phyllogens (3FLAG-PHYL RYD , PHYL OY or PHYL SY ) and yellow fluorescent protein (YFP)fused MTFs (AP1-YFP or SEP3-YFP) in N. benthamiana leaves, and 3FLAG-PHYL was immunoprecipitated by an anti-FLAG antibody.
AP1-YFP and SEP3-YFP were co-immunoprecipitated with 3FLAG-PHYL OY , but not with 3FLAG-PHYL SY .This result implies correspondence between MTF-binding affinity and MTF degradation activity in these phyllogens, as reported previously (Iwabuchi et al., 2020).In contrast, both AP1-YFP and SEP3-YFP were co-immunoprecipitated as efficiently with 3FLAG-PHYL RYD as with 3FLAG-PHYL OY , indicating that PHYL RYD retains affinity to non-PHYL RYD -degradable MTFs in planta.These results clarified that the low MTF degradation activity of PHYL RYD was not attributable to a defect in MTF-binding affinity.

| RAD23 recruitment ability of PHYL RYD
In addition to floral MTFs, phyllogen also binds to RAD23, a proteasomal shuttle protein (Kitazawa et al., 2022;MacLean et al., 2014).Y2H assays confirmed that PHYL RYD could interact with RAD23 of both A. thaliana and O. sativa in yeast cells (Table S1 and Figure S4).Kitazawa et al. (2022) proposed that the formation of the MTF/phyllogen complex triggers RAD23 recruitment in planta, resulting in the proteasomal degradation of the target MTF.Based on this model, we hypothesized that the low MTF degradation activity of PHYL RYD was caused by impaired RAD23 recruitment ability of the MTF/PHYL RYD complex.
To compare the RAD23 recruitment ability between AP1 (PHYL RYDdegradable MTF) and SEP3 (non-PHYL RYD -degradable MTF) when forming a complex with PHYL RYD , co-immunoprecipitation assays were performed in planta (Figure 5).AP1-YFP and myc-fused RAD23C (myc-RAD23C) were transiently expressed with and without 3FLAG-PHYL RYD in N. benthamiana leaves, and AP1-YFP was immunoprecipitated using an anti-green fluorescent protein (GFP) antibody.In the presence of 3FLAG-PHYL RYD , both 3FLAG-PHYL RYD and myc-RAD23C TA B L E 1 PHYL RYD interacts with non-degradable MADS box transcription factors (MTFs) in yeast.were co-immunoprecipitated with AP1-YFP (Figure 5a).This result indicates that the AP1/PHYL RYD complex can recruit RAD23C.In contrast, when SEP3-YFP was used instead of AP1-YFP, 3FLAG-PHYL RYD was efficiently co-immunoprecipitated with SEP3-YFP, whereas myc-RAD23C was not (Figure 5b).This result demonstrates that the SEP3/ PHYL RYD complex cannot recruit RAD23C, which is consistent with the inability of PHYL RYD to degrade SEP3 (Figure 3a).Notably, when PHYL OY was used instead of PHYL RYD , both the AP1/PHYL OY complex and the SEP3/PHYL OY complex were able to recruit RAD23C (Figure 5).

Oryza
Together, these findings show that RAD23 recruitment ability of the MTF/phyllogen complex differs depending on the combination of MTF and phyllogen and corresponds to the degradation activity.

| Novel mechanism for the target specificity of phyllogens in MTF degradation
The existing body of research on various effector proteins suggests that effector target specificity is often determined by its binding properties in relation to host proteins (Huang et al., 2021;Oikawa et al., 2020;Pecher et al., 2019;Tanaka et al., 2019).For phyllogen as well, several findings have indicated that the MTF-binding affinity of phyllogen determines its target specificity for MTF degradation (Iwabuchi et al., 2019(Iwabuchi et al., , 2020;;Liao et al., 2019;MacLean et al., 2014).
Phyllogen homologues that degrade floral MTFs (e.g., PHYL OY in Figure 6) first bind to a target MTF, and the MTF/phyllogen complex then recruit RAD23 to induce MTF degradation (Kitazawa et al., 2022).On the contrary, phyllogen homologues that cannot degrade floral MTFs (e.g., PHYL SY in Figure 6) do not bind to those MTFs (Iwabuchi et al., 2020).
The present study analysed PHYL RYD , a phyllogen homologue of RYD phytoplasma, and revealed a novel mechanism for the degradation target specificity of phyllogen in which the MTF degradation specificity is determined by RAD23 recruitment ability rather than MTF-binding affinity.PHYL RYD retains residues that are important for MTF binding (Figure 1) and binds to non-target MTFs that PHYL RYD is unable to degrade (Table 1 and Figure 4).Interestingly, although the complex of PHYL RYD and AP1 (PHYL RYD -degradable MTF) efficiently recruited RAD23, the complex of PHYL RYD and SEP3 (non-PHYL RYD -degradable MTF) could not (Figure 5).These results elucidated that the RAD23 recruitment ability of the MTF/ PHYL RYD complex differs according to the type of interacting MTF, resulting in a difference in degradation activity (Figure 6).Given that phyllogen homologues share the same mechanism for MTF degradation (Kitazawa et al., 2022), the finding that RAD23 recruitment ability determines the MTF degradation activity would be applicable to other phyllogens than PHYL RYD .Thus, the target specificity of phyllogen in MTF degradation is probably regulated by two steps: the MTF-binding affinity of phyllogen and the RAD23 recruitment ability of the resulting MTF/phyllogen complex (Figure 6).The discovery of this two-step target regulation mechanism also implies that caution is warranted when discussing the target specificity and functions of effector proteins solely on the basis of their binding affinity to host proteins.Kitazawa et al. (2022) showed that the MTF/phyllogen complex directly binds to RAD23 without ubiquitin, leading to the proteasomal degradation of host MTF in a ubiquitin-independent manner.This finding is noteworthy, as previous research has indicated that effector-mediated proteasomal degradation of host factors typically requires their ubiquitination (Ashida et al., 2014;Lin & Machner, 2017).To further elucidate this unique target degradation mechanism, the next step is to investigate how the MTF/phyllogen complex interacts with RAD23.Phyllogens recognize the K domain of MTF (MTF K ) and a ubiquitin-associated 2 (UBA2) domain of RAD23, a domain that originally binds to ubiquitin chains (Kitazawa et al., 2022).Because neither phyllogen nor MTF K

| Elucidating the molecular mechanism of RAD23 recruitment
shows structural similarity with ubiquitin, and no residues have been identified that are important for the interaction with UBA2, F I G U R E 4 PHYL RYD interacts with AP1 and SEP3 in planta.MTF-binding affinity of phyllogen homologues was examined by co-immunoprecipitation assay in planta.3FLAG-PHYL and MTF-YFP were co-expressed in Nicotiana benthamiana leaves by agroinfiltration.Total proteins were extracted 36 h post-infiltration (Input), and 3FLAG-PHYL was immunoprecipitated using an anti-FLAG antibody (IP by FLAG).Accumulation of 3FLAG-PHYL and MTF-YFP was evaluated by immunoblotting using anti-FLAG and anti-GFP antibodies, respectively.Coomassie brilliant blue (CBB) staining is shown as a loading control.
it is difficult to predict how the UBA2 domain binds to the MTF K / phyllogen complex.The results of this study revealed that SEP3/ PHYL RYD has a much lower affinity to RAD23C compared with the AP1/PHYL RYD and SEP3/PHYL OY complexes (Figure 5), which suggests that comparing the sequences and structures of AP1 K and SEP3 K , or those of PHYL RYD and PHYL OY , would help to identify the key residues or structures responsible for the interaction with RAD23 and shed light on how the MTF/phyllogen complex recruits RAD23.

| Importance of evaluating MTF degradation to analyse phyllogen functions
Several studies have raised the possibility that phyllogen may possess additional functions beyond its capacity to induce phyllody.Phyllogen expression has been shown to enhance the colonization of insect vectors, although the underlying molecular mechanism remains to be determined (Orlovskis & Hogenhout, 2016).Additionally, comprehensive Y2H assays have demonstrated that phyllogen can interact not only F I G U R E 5 AP1/PHYL RYD complex efficiently recruits RAD23C, but SEP3/PHYL RYD complex does not.(a) Co-immunoprecipitation assays to examine ternary interaction between AP1, PHYL RYD and RAD23C.AP1-YFP, 3FLAG-PHYL RYD and myc-RAD23C were coexpressed in Nicotiana benthamiana leaves by agroinfiltration.Total proteins were extracted 36 h post-infiltration (Input), and AP1-YFP was immunoprecipitated using an anti-GFP antibody (IP by GFP).Accumulation of AP1-YFP, 3FLAG-PHYL and myc-RAD23C was analysed by immunoblotting using anti-GFP, anti-FLAG and anti-myc antibodies, respectively.Coomassie brilliant blue (CBB) staining is shown as a loading control.(b) Co-immunoprecipitation assays using SEP3-YFP, 3FLAG-PHYL and myc-RAD23C.The experimental conditions were as described in (a).with floral MTFs but also with several other non-floral MTFs involved in diverse plant developmental processes (Correa Marrero et al., 2023;MacLean et al., 2014).Moreover, the binding affinity to the non-floral MTFs can differ among phyllogen homologues (Kitazawa et al., 2023).
However, whether phyllogen can degrade these non-floral MTFs remains to be clarified.This study elucidated that MTFs interacting with phyllogen are not necessarily degraded, emphasizing the importance of examining MTF degradation activity rather than MTF-binding affinity.Comprehensive MTF degradation assays will significantly advance our understanding of the functions of phyllogen.

| Functional characteristics of PHYL RYD
Previously characterized phyllogens belonging to the phyl-A, C and D groups (e.g., PHYL OY ) efficiently degrade AP1 and SEP1-4 and cause phyllody in A. thaliana, whereas phyllogens in the phyl-B group (e.g., PHYL SY ) do not degrade either AP1 or SEP1-4 and do not induce visible flower abnormalities (Iwabuchi et al., 2020).When compared to these phyllogens, PHYL RYD is unique in both the narrow range of target MTFs (Figure 3) and the induction of the intermediate phyllody phenotypes (Figures 2 and S2).
The intermediate phyllody observed in PHYL RYD -expressing A.
thaliana probably reflects its narrower degradation targets.AP1 gene encodes the only A-class MTF in A. thaliana, and ap1 single mutants show various abnormalities in sepals and petals but seldom induce stamen and pistil disorders (Chuang & Meyerowitz, 2000;Gregis et al., 2006).Similarly, PHYL RYD -expressing plants mainly exhibited abnormalities in sepals and petals, whereas stamens and pistils were less affected (Figure 2).This finding suggests that AP1 degradation could be involved in the induction of floral abnormalities by PHYL RYD .On the contrary, SEP1-4 genes encoding E-class MTFs in A. thaliana are functionally redundant, and the conversion of floral organs into sepalor leaf-like organs is observed only in triple and quadruple sep mutants (Ditta et al., 2004;Irish, 2017).Therefore, the compromised phyllodyinducing activity of PHYL RYD can be attributed to its low degradation ability for SEP3 and SEP4.Besides, floral MTFs are widely conserved in angiosperms (Irish, 2017), and phyllogens can degrade floral MTFs in various plant species (Kitazawa et al., 2017).Although the degradation activity of PHYL RYD for the floral MTFs of N. benthamiana was not examined in this study, the compromised phyllody-inducing activity of PHYL RYD observed in N. benthamiana (Figures 2 and S2) may reflect its narrow range of target MTFs, as observed in A. thaliana.

| Considerations on the function of PHYL RYD in rice
Certain phytoplasma strains lack a functional phyllogen due to the absence or pseudogenization of the phyllogen gene in the genome (Aurin et al., 2020;Iwabuchi et al., 2020;Maejima, Iwai, et al., 2014).
In contrast, PHYL RYD retains the full open reading frame, despite accumulating multiple mutations (Figure 1a).Therefore, there is an interest in the biological functions of PHYL RYD .The flowers and inflorescences of grass species including rice are completely different from those of other plant species such as A. thaliana and N.
benthamiana: one or several flowers lacking sepals and petals compose one small spikelet, and an aggregate of a substantial number of spikelets composes one ear (Yamaguchi & Hirano, 2006).Based on this characteristic, we propose two hypotheses regarding the biological implication of the narrower range of target MTFs of PHYL RYD .
One hypothesis is that PHYL RYD is gradually losing its function and may not significantly contribute to RYD phytoplasma infection.Su et al. (2011) reported that phytoplasmas infecting periwinkle plants accumulate to a greater extent in flowers with phyllody symptoms than in surrounding leaves; they proposed that phyllody might help to increase the phytoplasma population.However, a grass spikelet has no sepals or petals and is too tiny to significantly increase the phytoplasma population by inducing phyllody.In addition, no fulllength conserved phyllogen has been found in phytoplasma strains infecting cereal grasses, except for PHYL RYD (Iwabuchi et al., 2020;Zhu et al., 2017).These considerations suggest that gene loss or functional impairment of phyllogens may have occurred in grassinfecting phytoplasmas, possibly because phyllogens were dispensable due to the unique inflorescence architecture of their host plants.
An alternative hypothesis is that PHYL RYD selectively degrades certain Oryza MTFs that regulate inflorescence development.Among AP1-like Oryza MTFs, PHYL RYD degrades OsMADS14 and OsMADS15, but not OsMADS18 (Figure 3b).Despite the high degree of sequence similarity among these three Oryza MTFs, their functions are distinct.
OsMADS18 is expressed in most tissues and is involved in diverse developmental processes, including abscisic acid response, seed germination and flowering time (Wu et al., 2017).In contrast, OsMADS14 and OsMADS15 are mainly expressed in the inflorescence to specify the inflorescence meristem identity, and loss of their functions leads to spikelet sterility or failure to produce ears (Wu et al., 2017).As rice plants infected by RYD phytoplasma exhibit spikelet sterility and reduced ear numbers (Komori, 1965), PHYL RYD may contribute to these symptoms by degrading OsMADS14 and OsMADS15.Furthermore, rice mutants with spikelet sterility exhibit increased tillers, sustained high photosynthesis activity and delayed senescence (Kato et al., 2004(Kato et al., , 2006)).These secondary changes may enhance the fitness of RYD phytoplasma by improving the trophic conditions in sieve tissue and increasing the chance of transmission by its insect vector.
Further investigation into the biological functions of PHYL RYD in rice will advance our understanding of the functional diversification of the phyllogen family beyond the induction of phyllody symptoms.

| Materials
A. thaliana ecotype Col-0 was maintained in a growth chamber under 16-h light/8-h dark conditions at 22°C.N. benthamiana was grown under natural light conditions at 25°C before use and maintained in a

| MTF degradation assay
To examine the MTF degradation activity of phyllogen in N.
thaliana, 3FLAG-PHYL OY and 3FLAG-PHYL SY were constructed previously (Iwabuchi et al., 2019(Iwabuchi et al., , 2020)).Plasmid transformation and preparation of Agrobacterium suspensions were conducted as described above.Agrobacterium suspensions transformed with p19, myc-MTF and 3FLAG-PHYL (OD 600 = 1.0 for each) were mixed at a ratio of 1:10:1.For the mixture without 3FLAG-PHYL, an Agrobacterium-free infiltration buffer (Takahashi et al., 2006) was added instead of the 3FLAG-PHYL suspension.Mixtures with and without 3FLAG-PHYL were infiltrated on the right and left side, respectively, of the same leaf of a 4-week-old N. benthamiana plant.At 36 h after infiltration, leaf disks of the infiltrated area were collected and frozen immediately in liquid nitrogen.
Protein extraction and western blot analyses were carried out as described previously (Kitazawa et al., 2017).Proteins were detected by anti-myc (4A6; Millipore), anti-FLAG (F1804; Sigma-Aldrich) and anti-GFP (7.1 and 13.1; Roche) antibodies.For each combination of MTF and phyllogen, at least two replicates were prepared in each experiment and at least two independent experiments were conducted.

F
I G U R E 3 Degradation activity of PHYL RYD for several MADS box transcription factors (MTFs) is reduced.(a) The degradation activity of PHYL RYD for Arabidopsis MTFs.Each myc-fused MTF (myc-MTF) was transiently expressed with or without 3FLAG-fused PHYL RYD (3FLAG-PHYL RYD ) in Nicotiana benthamiana leaves by agroinfiltration.3FLAG-fused PHYL OY (3FLAG-PHYL OY ) was used as a positive control.Accumulation of myc-MTF and 3FLAG-PHYL was evaluated by immunoblotting using anti-myc and anti-FLAG antibodies, respectively.Membranes stained by Coomassie brilliant blue (CBB) are shown as loading controls.(b) The degradation activity of PHYL RYD for AP1-like and SEP-like Oryza MTFs.In addition to PHYL RYD , phyllody-inducing phyllogen PHYL OY and non-phyllody-inducing phyllogen PHYL SY were used.The experimental conditions were as described in (a).
The symbols indicate the growth of yeast co-expressing activation domain (AD)-fused MTF and DNA-binding domain (BD)-fused phyllogen on the selective media (−LWAH, −LWH + 3AT, −LWH and −LW): +++ the yeast grew on all the four media; ++ the yeast grew on −LWH + 3AT, −LWH and −LW; + the yeast grew on −LWH and −LW; − the yeast grew only on −LW.A higher number of + indicates stronger interaction between MTF and phyllogen in yeast, while − indicates that no significant interaction was detected.Raw data of yeast growth are shown in Figure S4.Several results were previously reported in a Iwabuchi et al. (2020), b Maejima, Iwai, et al. (2014), c Iwabuchi et al. (2019), and d Kitazawa et al. (2017).

F
I G U R E 6 MADS box transcription factor (MTF)-binding affinity and RAD23 recruitment ability cooperatively regulate target specificity of phyllogens.Phyllody-inducing phyllogens including PHYL OY first bind to target MTFs such as AP1 and SEP3, and then recruit RAD23 to induce MTF degradation.In contrast, non-phyllody-inducing phyllogens including PHYL SY have an impaired MTF-binding affinity and cannot efficiently degrade floral MTFs.PHYL RYD degrades a narrower range of floral MTFs than PHYL OY because RAD23 recruitment occurs only when PHYL RYD has recognized its target MTFs such as AP1.